Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) each utilizes four bases in a particular sequence to form genetic information. DNA uses adenine [A], guanine [G], thymine [T], and cytosine [C], while RNA uses A, G, C and Uracil (U). In both DNA and RNA, these bases are attached to a sugar-phosphate backbone having a 3′ end and an opposing 5′ end. When two single strands of DNA bind (associate) to form double-stranded DNA (hybridized or duplexed DNA), the 5′ to 3′ direction in one strand is positioned in a complementary manner, e.g., 180 degrees relative to the 5′ to 3′ direction of the other strand. A is complementary to T and G is complementary to C in DNA/DNA hybrids. A is complementary to U and G is complementary to C in DNA/RNA hybrids.
Amplification reactions are used to increase the number of DNA and/or RNA so that detection of specific sequences can be achieved. In certain types of amplification reactions, a relatively short sequence of DNA called a primer associates with a complementary sequence on the DNA template. A DNA polymerase uses deoxynucleoside triphosphates (dNTPs) to sequentially add nucleotides to the 3′ end of the primer (also referred to herein as “extension”). For extension to occur, a 3′ hydroxyl group is required on the ribose ring, which is the sugar moiety of the nucleic acid backbone. If the 3′ hydroxyl group of a nucleotide is replaced with a hydrogen atom, the nucleotide is known as a dideoxynucleotide (ddNTP), and cannot support attachment of an additional base during extension.
One of the drawbacks of certain amplification reactions is creation of “negative reaction products”. These negative reaction products have been found to be the result of the forward and reverse primers combining with one another to varying degrees in a template independent fashion. Although the precise mechanism for generation of this negative reaction product is not completely known, it is understood that certain reactions occur involving the primers in a forward-forward, forward-reverse or reverse-reverse configuration. These reactions can generate negative reaction products that can interfere with the accuracy of the detection process.